The microbial world is a microcosm whose activities are of central importance to the biosphere. Microbial products contribute to environment, plant, public, and soil health. There is a striking diversity of microorganisms in their ecological and physiological specialization. They have evolved to cope with and flourish in almost every niche, no matter how inhospitable. Microorganisms also form a range of associations with other microbes and with other plants and animals. They can be pathogens, parasites, symbionts, commensales and saprophytes, and thus, their ecological influence infiltrates into all trophic levels of life and gamut of possible ecosystems. Microbes have proved to be an exceptionally rich source of new products, and there is every indication that they will continue to be so in the future. Therefore, exploration of biodiversity for novel microbes that are of ecologically significance or are of economic value is of importance. This has prompted microbiologists to continue to search for novel useful microbes from sources that remain uncharacterized.
According to Hindu mythology as well as the Indian traditional medical practices (both the classical systems like Ayurveda and Siddha and the oral practices of the rural villagers) cow's milk has rejuvenatory health protecting and health promoting properties and hence has been said as the best one among vitalizers [Caraka-Samhita, Editor-translator P. Sharma, Chaukhambha Orientalia, Varanasi, India, volume 1, p. 213 (1981); P. Pushpangadan, Ethnobiology in India: A status report, Ministry of Environment and Forests, Government of India, New Delhi (1994); P. Pushpangadan, All India Coordinated research project on ethnobiology: Final technical report 1982–1998, Ministry of Environment and Forests, Government of India, New Delhi (1998)].
Milk may be defined as the normal secretion of the mammary gland of a mammal. Milk as it is secreted by the gland of mammals is free of microorganisms. However, microorganisms associated with the teat move up the teat canal and into the interior of the udder [J. C. Olsen and G. Mocquot. Milk and milk products. In: International commission on microbiological specifications for foods. Microbial ecology of foods. Food commodities. Vol. 2. New York: Academic Press (1980) pp. 470–486]. This causes even aseptically drawn milk to contain microorganisms, mostly bacteria. Bacteria in aseptically drawn milk are usually limited in number and include mostly micrococci, lactococci, staphylococci, streptococci, and bacillus [F. L. Bryan, Journal of Food Protection, Volume 46, pp. 637–649 (1983); R. A. Ledford. Raw milk and fluid milk products. In: Applied dairy microbiology. Eds. E. H. Marth and J. L. Steele, New York: Marcel Dekker, Inc. (1998) pp. 55–64].
It has been known for more than four decades that many of the bacteria that occur commonly in milk find it a relatively unfavorable medium and it would thus appear that milk has pronounced selective properties [T. Gibson and Y. A. Abd-El-Malek, Canadian Journal of Microbiology, Volume 3, pp. 203–213, (1957)]. Thus the bacterial flora that has invaded the teat and/or udder must have the ability to survive and multiply under these sub-optimal conditions. Therefore, work on the milk described in this application pertains to bacterial flora persisting in the teat and/or udder, which have gained entrance into the aseptically drawn milk, in our attempt to search for novel microbes, from an ecological niche that remains uncharacterized.
Improving soil fertility is one of the most common tactics to increase agricultural and forest production. We have isolated bacteria beneficial to plants from cow's milk. Inoculation of seeds or soil with beneficial microorganisms for crop improvement has been practiced for a number of years. A variety of mechanisms have been identified as being responsible for such plant growth promoting activity. For example, certain microorganisms indirectly promote plant growth by inhibiting the growth of deleterious microorganisms; or directly enhance plant growth by producing growth hormones; and/or by assisting in the uptake of nutrients by the crops, e.g., phosphorus (P) [C. S. Nautiyal et al., FEMS Microbiology Letters, Volume 182, pp. 291–296 (2000)].
However, a major factor in the unsuccessful commercialisation of bioinoculants has been the inconsistency of field test results as their establishment and performance are severely effected by environmental factors especially under stress conditions encountered in soil e.g., salt, pH, and temperature [C. S. Nautiyal et al., FEMS Microbiology Letters, Volume 182, pp. 291–296 (2000)]. Therefore, it would be desirable to provide stress tolerant bacterial strains as bioinoculants [C. S. Nautiyal, Biocontrol of plant diseases for agricultural sustainability. In: Biocontrol potential and its exploitation in sustainable agriculture. Volume 1, Eds. R. K. Upahyay, K. G. Mukerji, and B. P. Chamola, Kluwer Academic/Plenum Publishers, New York (2000) pp. 9–23]. Plant growth promoting microorganisms include but are not limited to Rhizobium, Pseudomonas, Azospirillum, and Bacillus etc. [A. K. Saxena et. al., Bacterial biocontrol agents and their role in plant disease management. In: Biocontrol potential and its exploitation in sustainable agriculture. Volume 1, Eds. R. K. Upadhyay, K. G. Mukerji, and B. P. Chamola, Kluwer Academic/Plenum Publishers, New York (2000) pp. 25–37].
Usefulness of B. subtilis as a source of an antagonist for plant pathogenic fungus Sclerotium rolfsii is well known [P. Broadbent et al., Australian Journal of Biological Sciences, Volume 24, pp. 975 (1971)]. Baker et al. [Phytopathology, Volume 73, 1148–1152 (1983)] also report on use of B. subtilis as a biocontrol agent of fungal plant pathogens. Pusey et al. [Plant Disease, Volume 72, pp. 622–626 (1988)] and P. L. Pusey [U.S. Pat. No. 5,047,239] disclosed control of post harvest fruit rot using B. subtilis. S. D. Heins et al. [U.S. Pat. No. 6,103,228] have disclosed methods of protecting or treating plants from fungal and bacterial infections and corn rootworm infestations using B. subtilis. 
B. lentimorbus is the causative agent of milky disease in Japanese beetle and related scarab larvae [K. E. Rippere et al. International Journal of Systematic Bacteriology, Volume 48, pp. 395–402 (1998)], and therefore is used for the biocontrol of larvae of certain insects [R. E. Gordon et al., The genus Bacillus, Agriculture handbook no. 427, United States Department of Agriculture, U.S. Government printing office, Washington D.C. (1973)]. B. lentimorbus has also been used to increase the production of fish [W. T. Logan et al., U.S. Pat. No. 5,746,155] and to treat poultry litter [W. T. Logan et al., U.S. Pat. No. 6,017,525].
For propagating bacteria commonly used carriers for commercial inoculants are vermiculite, charcoal, caboxymethyl cellulose, peat, perlite, polyvinyl-pyrrolidone, and talc. Press mud, a “waste” product obtained during sugar manufacture, has also been used as a carrier for Azotobacter chroococcum and Rhizobium japonicum [K. S. Jauhri, Indian Journal Agriculture Research, Volume 24, pp. 189–197 (1990)]. Press mud, like any other organic material, affects the physical, chemical and biological properties of the soils. It also helps to increase water stable aggregates in soils. It can be composted with distillery spent wash and utilized as a better organic material than press mud alone [D. P. Yadav. Recycling of sugar factory press mud in agriculture. In: Recycling of crop, animal, human, and industrial wastes in agriculture. Ed. H. L. S. Tandon, Fertilizer development and consultation organization, New Delhi (1995) pp. 91–108]. Agricultural and environmental industries would therefore clearly benefit from a simple, less expensive method of making microbial inoculants for plants, seeds and soil.
While work on the microbiology of milk so far has been on psychrotrophic bacteria because of their importance in milk and dairy products [M. A. Cousin. Journal of food protection. Volume 45, pp. 172–207 (1982); R. A. Ledford. Raw milk and fluid milk products. In: Applied dairy microbiology. Eds. E. H. Martha and J. L. Steele, New York: Marcel Dekker, Inc. (1998) pp. 55–64], no bacterial strain has been previously found from the milk of cows which has the ability to control phytopathogenic fungi, promote plant growth, provide tolerance for abiotic stresses, and solubilize phosphate under abiotic stress conditions.
India is one of the few countries in world that has contributed richly to the international livestock gene pool and improvement of animal population in the world. Cattle and buffalo contribute nearly 15% of the gross national income. The country possesses 23% of the world bovine population. “Cattle” is a common term for the domesticated herbivorous mammals that contribute to genus Bos, of the family Bovidae. Modern cattle are divided into two species: B. taurus, which originated in Europe and includes most modern breeds of dairy and beef cattle, and B. indicus, which originated in India and is characterized by a hump and the withers. The latter are now widespread in Africa and Asia, with lesser numbers imported to North America (primarily in the southern U.S.), Central America, and Northern and Central America.
Dairy cattle are those breeds that have been developed primarily to produce milk. In North America the major breeds of dairy cattle are the Holstein-Friesian, Ayrashire, Brown Swiss, and Jersey. Among the major dairy breeds of B. indicus found primarily in India are the Gir, Hariana, Red Sindhi, Tharparker, and Sahiwal. By far Sahiwal is the best breed of the subcontinent. It is comparatively a heavy breed with a symmetrical body and loose skin, when compared with Red Sindhi that it closely resembles. The animals are usually long and fleshy and with a heavier build. The color is redish dun or pale red, sometimes fleshed with white patches. A number of herds of this breed are maintained in India. The milk yield ranges from 1400 to 2500 kg. The heritability of this trait is 0.2 to 0.3. The age at first calving ranges from 37 to 48 months and the calving interval is from 430 to 580 days. Sahiwal is one of the most popular breeds of the subcontinent. It has been exported to Sr. Lanka, Kenya and many countries in Latin America and the West Indies where a new breed called Jamaica Hope has been evolved out of Sahiwal and Jersey crossbreeds [P. N. Bhat, Handbook of Animal Husbandry, Directorate of Publication and Information on Agriculture, Krishi Anusandhan Bhawan, Pusa, New Delhi (1997)].
Accordingly, there has been no clear indication heretofore that any bacteria isolated from cows might act as a biocontrol agent, and certainly no showing of direct, bacterial-mediated stimulation of plant growth per se. Nevertheless, a bacterial strain capable of promoting plant growth, tolerance for abiotic stresses, and that solubilizes phosphate under abiotic stress conditions, if one were isolated, could find immediate application, e.g., in soils affected by phytopathogens, poor availability of nutrients like phosphorus, and environment stresses etc. Additionally, no procedure for the selection of such bacterial strain has been reported. We have found by direct comparison on a variety of plant types that the unique combination of selected bacterial strains of the invention is effective in the enhancement of plant growth and health.
The present invention relates to novel strains of bacteria isolated from cows which have the ability to control phytopathogenic fungi, promote plant growth, improve tolerance for abiotic stresses, solubilize phosphate under abiotic stress conditions, and a method for the selection of these strains.